Microfluidic systems and methods of transport and lysis of cells and analysis of cell lysate

ABSTRACT

Microfluidic systems and methods are disclosed which are adapted to transport and lyse cellular components of a test sample for analysis. The disclosed microfluidic systems and methods, which employ an electric field to rupture the cell membrane, cause unusually rapid lysis, thereby minimizing continued cellular activity and resulting in greater accuracy of analysis of cell processes.

[0001] This invention was made with Government support under contractDE-AC05-00OR22725 awarded by the United States Department of Energy toUT-Battelle, LLC. The Government has certain rights in this invention.

FIELD OF THE INVENTION

[0002] This invention relates to molecular biology, and in particular,to the use of an applied electric field in a microfluidic system for themanipulation of biological samples comprising cells and cell lysate(s)for subsequent analysis.

BACKGROUND OF THE INVENTION

[0003] Interest in microfabricated devices for chemical sensing andanalysis has grown substantially over the past decade, primarily becausethese miniature devices have the potential to provide informationrapidly and reliably at low cost. Microchips fabricated on planarsubstrates are advantageous for manipulating small sample volumes,rapidly processing materials, and integrating sample pretreatment andseparation strategies. The ease with which materials can be manipulatedand the ability to fabricate structures with interconnecting channelsthat have essentially no dead volume contribute to the high performanceof these devices. See, for example U.S. Pat. Nos. 5,858,195 and6,001,229, which are commonly owned with this application. To carry outa complete analysis, many different kinds of functional elements can bedesigned and integrated on microchips. These elements include filters,valves, pumps, mixers, reactors, separators, cytometers and detectors,which can be operatively coupled together under computer control toenable the implementation of a wide range of microchip-based analyses.

[0004] One area of particular interest is the analysis of cells and cellpopulations. At present most techniques for cellular analysis dependupon pooling a population of cells to obtain a large enough quantity ofanalyte for detection. Pooling of cells, however, obscures any variationin analyte concentration from cell to cell. For many studies averageanalyte values across a large population of cells will be acceptable;however, for the study of processes such as carcinogenesis, the abilityto quantitate analytes in individual cells is required so that raremutations in cells, which lead to drastic changes in cell metabolism andprogression to cancer, can be detected.

[0005] With the advances in Capillary Electrophoresis over the last twodecades the quantitation of analytes in individual cells has becomefeasible albeit slow because of the intensive manual manipulations whichhave to be performed. The potential to automate and integrate celltransport, manipulation and lysis with separation and analyte detectionmake microfluidic devices a desirable platform for performing highthroughput screening of individual cells from large populations. A keystep in integrating cell handling with analyte detection andquantitation is providing a method of cell lysis which is rapid andgenerates small axial extent plugs for subsequent analysis. Because theresolution or separation between any pair of compounds can bedetrimentally affected by long injection plug lengths, small axialextent plugs are important for a successful separation. It should benoted, however, that while small axial extent plugs are desired, thecontents of the cell after lysis should be spread over several cellvolumes so that reaction pathways within the cell are terminated andproteolytic enzymes released from vesicles during lysis are sufficientlydiluted. This will prevent the possible digestion of proteins ofinterest.

[0006] The use of an applied electric field for cell lysis is known. Forexample, the lysis of erythrocytes in suspension by pulsed electricfields has been reported both for bovine (Sale and Hamilton, “Effects ofHigh Electric Fields on Microorganisms III, Lysis of Erythrocytes andProtoplasts”, Biochim et Biophys Acta, 163:37 (1967)) and humanerythrocytes (Kinosita and Tsong, “Voltage-Induced Pore Formation andHemolysis of Human Erythrocytes”, Biochim et Biophys Acta, 471:227(1977); and Kinosita and Tsong, “Hemolysis of Human Erythrocytes by aTransient Electric Filed”, Proc Nail Acad Sci. , 74:1923 (1977)). Thesereports indicate that applied electric fields resulting in cellulartransmembrane potentials on the order of 1 Volt can result in lysis oferythrocytes. However, these previously reported cell lysis techniquesutilizing an electric field are typically carried out in a macroscaledevice, rather than a microchip device. Consequently, such techniquesare lacking in certain respects. Specifically, the conditions employedin macroscale electric cell lysis devices do not consistently releaserelatively high molecular weight nucleic acid molecules, because suchmolecules do not readily pass through the pores created in the cellmembrane by this lysis technique. Also, the existing macroscale,electric lysis devices function as stand-alone units, thus precludingintegration of cell manipulation and lysis with separation and analysisof cell lysate in a unitary device.

[0007] Other methods of cell manipulation and/or lysis on microscaledevices have been proposed. See, for example, U.S. Pat. Nos. 4,676,274and 5,304,487. Chemical lysis on a microchip device has beendemonstrated by mixing a surfactant, i.e. sodium dodecylsulfate, withcanine erythrocyte cells. (Li and Harrison, Anal. Chem., 69:1564-1568(1997)). This report indicates that the cells were lysed in under 0.3sec. No subsequent analysis of the cellular contents, however, wasreported. Single erythrocytes have been lysed and the cell contentsseparated using two capillaries across which an electric field isapplied. A gap of about 5 μm is provided between the two capillaries. Asintact cells pass through the gap, they are lysed and the contents ofthe lysed cells are transported to the second capillary for separation.The lysis is presumed to be caused by the mechanical shear stressesinduced by the change in electric field strength between the capillariesand the gap region. The gap region is considerably larger in crosssectional area, so that the field strength and, therefore, cell velocityis lower than in the capillaries. See, Chen and Lillard, Anal. Chem.,73:111-18 (2001).

[0008] Cell lysis can be considered an extreme form of cell membranepermeablization (poration). Optoporation has been carried out usinghighly focused light from a pulsed laser (Soughhayer et al., Anal. Chem.72(6):1342-1347 (2000)). The laser is focused near the cell at theaqueous/glass interface. When the laser is pulsed, a stress (shock) waveis generated which transiently permeablizes the cell. This technique hasbeen shown to be capable of lysing cells, as well. The occurrence ofcell lysis or poration only is a function of the cell's distance fromthe laser focal spot.

[0009] Although the above-mentioned cell lysis techniques of the priorart are useful for certain applications, there exists a need in the artfor microchip-based cell manipulations and lysis which is sufficientlyrapid to minimize continued cellular activity after lysis, thusproducing greater accuracy in the analysis of cell processes.

SUMMARY OF THE INVENTION

[0010] In accordance with one aspect of the present invention, there isprovided a method of releasing the intracellular contents of at leastone cell of a cell-containing fluid sample for analysis. The method ofthe invention comprises providing a substrate having a microchannelstructure which includes one or more microchannel(s). An electric fieldis generated from a source of electric potential and applied in aspatially defined region of the aforementioned microchannel, whichfunctions as a cell lysis region. The strength of the applied electricfield is adequate to induce cell lysis. At least one cell of the fluidsample is positioned in the cell lysis region for a time sufficient torelease the intracellular contents into the fluid sample. The releasedintracellular contents form an analyte plug of narrow axial extent inthe microchannel.

[0011] According to another aspect of the invention, there is provided amicrofluidic system for transport and lysis of at least one cell of acell-containing fluid sample. The microfluidic system of this embodimentof the invention comprises a source of electric potential and a solidsubstrate including one or more microchannel(s) with a longitudinalaxis, and a cell lysis region between first and second electricalcontacts positioned adjacent (i.e. on or in close proximity to)microchannel wall portions on different sides of the longitudinal axis.The electrical contacts, which are spatially separated by the cell lysisregion and electrically isolated from one another, are connected to asource of electric potential, which is operative to apply an electricfield to the cell lysis region, transverse to the fluid sample flowpath, within the microchannel space between the electrical contacts. Thesystem also includes means for transporting the cell-containing fluidsample along the aforementioned microchannel.

[0012] According to a further aspect of this invention, there isprovided a microfluidic system for transport and lysis of at least onecell of a cell-containing fluid sample and separation of itsintracellular content. This system comprises a solid substrate havingone or more microchannel(s) disposed therein, which includes a celltransport segment and a separation segment with first and second endportions. A first and a second electrode are provided along theaforementioned microchannel, intermediate the transport segment and theseparation segment, and are spatially separated from one another,defining a space in the microchannel between them that serves as a celllysis region. The electrodes are connected to a source of electricpotential to apply an electric field to the cell lysis region. Thissystem further includes means for flowing the cell-containing fluidsample through the aforementioned microchannel and means for applying anelectric potential difference between the first and second separationsegment end portions for effecting separation of the intracellularcontents of lysed cells.

[0013] The microfluidic system and methods of this invention enableexamination of the contents of single cells with high throughput.Consequently, this invention is expected to have considerable utilityfor facilitating research in the life sciences, and especially thepharmaceutical industry. For example, implementation of this inventionin the pharmaceutical industry could expedite screening of cellularresponses to large combinatorial libraries of potential novel drugs. Inaddition, the present invention can be used to advantage for the studyof carcinogenesis or oncogenesis by assisting in the detection of rarecell mutations at an early stage, which is considered essential to thesuccessful treatment of various forms of cancer. This invention may alsobe used to further elucidate metabolic pathways in cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 includes five (5) separate diagrammatic illustrations ofmicrochannel structures of a microfluidic system embodying the presentinvention. In FIG. 1A, cell lysis is effected in the applied electricfield generated between a first electrical contact integrated into themicrochannel and a second electrical contact positioned at or near themicrochannel terminus, e.g. in a reservoir. FIG. 1B shows a differentmicrochannel structure, in which a bridging membrane is included toprovide electrical contact between the vertical and horizontal channels,without creating significant concurrent fluid flow. FIGS. 1C-1E showmicrochannel structures which have, as a common feature, a pair ofelectrical contacts which generate an electric field, causing cell lysisin the microchannel space between the electrical contacts. Themicrochannel structure of FIG. 1C also includes a separate means forinitiating a cell lysis electric field, recording a time mark toindicate the start of a separation process, or controlling fluid sampletransport, such as starting and/or stopping sample flow.

[0015]FIG. 2 includes three (3) separate diagrammatic illustrations ofmicrochannel structures of a microfluidic system according to thepresent invention, including auxiliary channel segments which intersectwith, and enable introduction of various agents into the microchannel,wherein cell transport and lysis occur. FIG. 2A shows a microchannelstructure with a single auxiliary or side channel forming a tee-shapedintersection with the transport/lysis microchannel. FIG. 2B shows across design including two (2) side channels intersecting thetransport/lysis microchannel on opposite sides thereof. In themicrochannel structure illustrated in FIGS. 2A and 2B, the cell isexposed to the electric field upon entering the intersection. FIG. 2Cshows an alternative cross microchannel structure designed for injectionof a dilution buffer into the transport/lysis microchannel in order tominimize localized heating of the fluid sample comprising physiologicalbuffer, while allowing maintenance of a high electric field in thechannel segment downstream of the intersection for cell lysis andsubsequent separation.

[0016]FIG. 3 provides two (2) diagrammatic illustrations of microchannelstructures of a microfluidic system according to the present invention,in which a cross-section dimension of the transport/lysis microchannelis varied, either by expansion (FIG. 3A) or constriction (FIG. 3B), tochange the field strength to which the cell is exposed.

[0017]FIG. 4 includes two (2) diagrammatic illustrations of microchannelstructures of a microfluidic system according to the present invention,which are designed to avoid possible interference with hydraulicpressure operating on sample fluid in the transport/lysis microchannel,due to evolution of gas at an electrode or electrodes positioned in ornear the transport/lysis microchannel, e.g. in a channel or fluidreservoir where pressure is applied. This may be accomplished by placingthe electrodes at the end of an auxiliary channel, as see in FIG. 4A, orin a channel connected to transport/lysis microchannel via a bridgingmembrane, as see in FIG. 4B.

[0018]FIG. 5 is a diagrammatic illustration of cell lysis and lysateseparation performed in a microfluidic system according to the presentinvention, having an electric field transverse to the direction of flowof the cell-containing fluid sample. FIG. 5A shows the basicmicrochannel structure with electrodes placed at the termini (e.g., influid reservoirs) of side channels that intersect the transport/lysismicrochannel. The arrow indicates the direction of pressure driven flowof the fluid sample. FIG. 5B shows the cell being transported toward theintersection via the pressure driven flow. FIG. 5C illustrates lysis ofthe cell at the intersection and release of the intracellular content(s)which begin migrating along the right side channel. FIG. 5D showschemical separation of the cell lysate along the right side channel.

[0019]FIG. 6 is a diagrammatic illustration of a microchannel structureof a microfluidic system combining the features of flow cytometry tospatially confine and detect the presence of a cell with the feature ofcell lysis, as illustrated in FIG. 5, above.

[0020]FIG. 7 includes a series of CCD camera images of cells uponexposure to an applied electric field in a microfluidic system inaccordance with the present invention. Cells are lysed in a pulsedelectric field, as seen in FIG. 6A (frames 1-7), but remain intact,albeit distorted, when the electric field is not applied, as seen inFIG. 6B (frames 1-6).

[0021]FIG. 8 is an electropherogram that depicts the analysis viaelectrophoresis of intracellular contents following cell lysis, in themanner illustrated in FIG. 5.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The microfluidic devices and systems described herein can be madeusing various microfabrication techniques, as described in theaforementioned U.S. Pat. Nos. 6,001,229, issued Dec. 14, 1999 and6,033,546, issued Mar. 7, 2000, both to J. M. Ramsey, as well as in U.S.patent application Ser. No. 09/244,914, filed Feb. 4, 1999 in the namesof S. C. Jacobson et al. The devices and systems of this invention mayalso include means to induce pressure electrokinetically, for effectingmaterial transport, as described in U.S. Pat. Nos. 6,110,343 and5,231,737 to R. Ramsey and J. M. Ramsey. The entire disclosures of thelast mentioned patents and application are incorporated by reference inthe present application.

[0023] In embodiments of the invention employing one or more reservoirsfor delivery or collection of a test sample, diluent, reagent or thelike, electrode placement in a reservoir is done in the manner describedin the patents and patent application referred to immediately above.Integration of an electrode into a microchannel structure, which is afeature of certain embodiments of this invention, may be done bydepositing a thin metal film (˜100 nm) of either Ti or Cr as an adhesionlayer on a glass substrate followed by a thin metal film of gold (˜300nm). Photoresist is then spun on the metallized substrate and a mask ofthe desired electrode pattern is placed on top of the photoresistcovered metallized substrate. The areas of the photoresist exposed,i.e., not covered by the electrode pattern, are removed, and the metaletched away. See, e.g., McKnight et al., 2001. Anal. Chem 73, 4045-4049.This leaves the glass substrate with a finished electrode pattern, ontowhich a poly(dimethylsiloxane) (PDMS) substrate with channels moldedtherein can be bonded. The bonding can be accomplished by either simplecontact bonding or through covalent bonding generated by exposing theglass and PDMS substrates to an oxygen plasma and then bringing themquickly into contact with each other. See, e.g., Duffy et al., 1998,Anal. Chem. 70, 4974. The entire disclosures of the last-mentioned twoliterature references are incorporated by reference in the presentapplication. See also, the aforementioned U.S. patent application Ser.No. 09/244,914.

[0024] The microfluidic devices and systems used in practicing thisinvention can be made out of a variety of substrate materials, includingglass, fused silica and various polymeric materials, such as PDMS orcombinations of such materials, as described above.

[0025] A number of different microchannel structures on microfluidicdevices embodying the present invention are diagrammatically shown inFIG. 1. The microchannel structure 10, as shown in FIG. 1A employs anelectrode 12 integrated into microchannel 14, together with anotherelectrode 16, preferably positioned at or near the end of channel 14, toapply an electric field of sufficient strength in cell lysis region 17to lyse cell 11. The substrate of this device is advantageouslyfabricated out of PDMS, which allows gaseous electrolysis productsgenerated at the integrated electrode 12 to be removed by rapiddiffusion through the PDMS, as disclosed in the aforementioned U.S.patent application Ser. No. 09/244,914. In this and other embodimentsthat include an in-channel electrode, the electrode may be integrated,in part, on the cover plate of the device. The electric field isgenerated by a source of electric potential 19, which is connected toelectrodes 12 and 16. The arrow in microchannel 14 indicates thedirection of sample flow as a cell-containing fluid sample traverses theflow path along the microchannel.

[0026] In the microchannel structure 20 of FIG. 1B, a bridging membrane23, as disclosed in the aforementioned U.S. patent application Ser. No.09/244,914, may be used to provide electrical contact between channel 24and channel 28, and thereby minimize concurrent fluid flow. Electrode 22is preferably placed at or near the end of channel 24, e.g. in areservoir (not shown) in fluid communication with channel 24, andelectrode 26 is similarly placed in channel 28. The electrodes areconnected to a source of electrical potential 29, which is operative toapply an electric field to cell lysis region 27, thus inducing lysis ofcell 21. The arrow in microchannel 24 has the same significance asmentioned above, with reference to FIG. 1A.

[0027] In the microchannel structure 30 shown in FIG. 1C, microchannel34 has four electrodes 32 a, 32 b, 36 a, 36 b. Several modes ofoperation are possible with this design. In a first mode of operation,electrodes 32 a and 32 b with an electric potential applied are used forcell lysis, and electrodes 36 a and 36 b are not needed. The presence ofa cell 31 in zone 37 a can be determined by a change in conductivity andcan be used to trigger events such as the application of a cell lysiselectric field in zone 37 a between electrodes 32 a and 32 b, therecording of a time mark to indicate the start of a separation process,or fluid control actions, such as starting or stopping flow to controltransport of the triggering cell or other cells. Separation and/oranalysis of the contents of cell 31 may be performed in the microchannelspace downstream of zone 37 a. In a second mode of operation, electrodes32 a and 32 b can be used for cell lysis in zone 37 a, and electrodes 36a and 36 b can be used for electrokinetic separations in zone 37 b. Thisallows field strengths of different magnitudes to be used for cell lysisin zone 37 a and separation in zone 37 b. Also, electrodes 32 b and 36 acan be common for this mode of operation. A third mode of operation useselectrodes 32 a and 32 b to detect the presence of a cell 31 in zone 37a by a change in conductivity. This change in conductivity can be usedto trigger the cell lysis electric field in zone 37 b between electrodes36 a and 36 b. Also, electrodes 32 b and 36 a can be common for thismode of operation. Electrodes 32 a, 32 b, 36 a, 36 b can be positionedat, in, or near microchannel 34 to effect the above modes of operation.The pairs of electrodes 32 a and 32 b and 36 a and 36 b are connected tosources of electric potential 39 a and 39 b, respectively, to generatethe appropriate electric fields. These electric fields can include DCand/or AC components. Also depicted is an alternative method fordetermining the presence of a cell in microchannel 34 using an opticalprobe. A light source 35 a can be directed toward microchannel 34preferably either above (as depicted with dashed line with arrow) or inzone 37 a. If a cell 31 is present, scattered light includingfluorescence is detected by detector 35 b (depicted by dashed line witharrow). In this way, a cell lysis electric field can be generated inzone 37 a between electrodes 32 a and 32 b, or other events as discussedabove. An optical probe or conductivity measurement to detect thepresence of the cell in a microchannel can be used in conjunction withany of the lysis techniques described in this application. The solidline with arrow in microchannel 34 has the same significance asmentioned above, with reference to FIG. 1A.

[0028]FIG. 1D shows a microchannel structure 40 including microchannel44 having a longitudinal axis with a first side wall portion 44 a on oneside of the axis, a second side wall portion 44 b on the other side ofthe axis and a cell lysis region 47 defined by the microchannel spacebetween first and second electrodes 42, 46 positioned at or near sidewall portions 44 a and 44 b, respectively. The electrodes are connectedto a source of electric potential 49, which is operative to apply anelectric field to cell lysis region 47, thus inducing lysis of cell 41.The arrow shown in microchannel 44 has the same significance asmentioned above, with reference to FIG. 1A.

[0029] The microchannel structure 50 shown in FIG. 1E is quite similarto that of FIG. 1D, including microchannel 54 with first and secondelectrodes 52, 56 positioned at or near first and second microchannelside wall portions 54 a and 54 b, defining a cell lysis region 57 and asource of electric potential 59, which is connected to the electrodes togenerate an electric field which effects lysis of cell 51 in the celllysis region. The microfluidic device of FIG. 1E, in contrast to that ofFIG. 1D, has electrical contacts configured to prolong the residencetime of the cell(s) in the electric field. The arrow shown inmicrochannel 54 has the same significance as mentioned above, withreference to FIG. 1A. The embodiments of the invention described withreference to FIGS. 1A-1E can be beneficially used for analyses ofcellular contents involving any number of chemical separationtechniques, such as electrophoresis or chromatography.

[0030] In all of the embodiments of this invention shown in FIGS. 1A-1E,electrodes or bridging membranes can be used to make electrical contactwith microchannels.

[0031] The electrode configurations in FIGS. 1D and 1E can also be usedto detect the presence of a cell by measuring a change in conductivity.This change in conductivity can be used to trigger the cell lysisprocess. Additional electrodes configured similarly can be added to themicrochannel to segregate the processes of detecting the presence of thecell and lysing the cell. Operation would be similar to that describedfor above FIG. 1C.

[0032] In the microchannel structures provided in the devices shown inFIGS. 1C, 1D and 1E, cell lysis can be effected by having the electricalcontacts in close proximity to the microchannel, but not in physicalcontact with the fluid sample, and applying an AC potential.

[0033] In the microchannel structure 60 of FIG. 2A, microchannel 64, inwhich cell transport and lysis occurs, is joined by side channel 65,forming a tee-shaped intersection 63. One electrode 62 is positioned ator near the terminus of channel 64, whereas another electrode 66 issimilarly positioned in microchannel 65. The electrodes are connected toa source of electrical potential 69. This electrode arrangementeffectively establishes an electric field comprising the cell lysis andseparation region 67 between intersection 63 and the aforementionedterminus of channel 64. Cell 61 is exposed to the electric field as itenters intersection 63.

[0034] An operational advantage of this embodiment of the invention isthat a fluid can be introduced through side microchannel 65, e.g., froma reservoir (not shown) in fluid communication with side microchannel65, to alter the buffer composition in transport/lysis microchannel 64.In this way, the conductivity of the cell-containing fluid sample may bereduced, thus diminishing the risk of Joule heating of the fluid sample,which can occur when a high electric field is applied to cell-containingsamples prepared using conventional physiological buffers, e.g.,phosphate buffered saline (PBS). The inclusion of an intersecting sidechannel also enables the addition of a chemical lysing or solubilizationagent, e.g., a surfactant, to the fluid sample in transport/lysismicrochannel 64. In addition, the rate of flow of material along eachintersecting microchannel may be adjusted to provide various mixingratios, either by changing the flow resistance in the channels, byaltering the applied pressure or applied field strength.

[0035] The embodiment of FIG. 2B is analogous to that of FIG. 2A, inthat it is in the form of a microchannel structure 70, including atransport/lysis microchannel 74 which is intersected by a side channel.In this case, however, a pair of opposed side channels 75 a and 75 bform a cross-intersection 73 with microchannel 74. Each side channel hasan electrode 72 a, 72 b positioned at or near the terminus thereof,e.g., in a reservoir (not shown) containing a dilution buffer,solubilization agent, chemical lysis agent, or the like. Anotherelectrode 76 is positioned at the lower end of microchannel 74 and isconnected to two (2) separate sources of electric potential 79 a, 79 b,which are also connected to electrodes 72 a apt and 72 b, thusestablishing an electric field comprising cell lysis and separationregion 77, within the microchannel space between the intersection 73 andelectrode 76. Cell 71 is exposed to lysis conditions upon enteringcross-intersection 73. This embodiment provides all of the operationaladvantages of the microfluidic device shown in FIG. 2A and additionallyenables cells to be exposed even more quickly and symmetrically to theeffects of any chemical lysis agent or lipid membrane solubilizationagent. Although two sources of electric potential, are depicted in FIG.2B, only one source is needed to effect lysis, e.g. 79 a or 79 b, withelectrode 72 a and/or 72 b connected to the single source of electricalpotential.

[0036] Another approach to overcoming the high conductivity problemexperienced when using cell-containing fluid samples, e.g. PBS, isillustrated in the microchannel structure 80 of FIG. 2C. Thisembodiment, like that of FIG. 2B, includes a microchannel 84, with apair of opposing side channels 85 a and 85 b in a cross design. Thefunction of the side channels is to introduce dilution buffer, chemicallysing agent or a solubilization agent into transport/lysis microchannel84. One electrode 82 is positioned downstream of intersection 83, formedbetween side microchannels 85 a, 85 b and transport/lysis microchannel84, e.g., in a waste reservoir (not shown) in fluid communication withmicrochannel 84. Another electrode 86 is positioned at or near theterminus of auxiliary channel 88. The electrodes are connected to asource of electric potential 89 to generate an electric field comprisingcell lysis region 87 within the lower portion of microchannel 84. Usingthis design, a dilution buffer or other fluid can be introduced intomicrochannel 84 through side channels 85 a, 85 b or both of them, whichdilutes the cell-containing fluid samples sufficiently such that a highelectric field may be applied in the lower segment of microchannel 84. Amicrochip design having only one side channel, 85 a or 85 b, can achievea similar result.

[0037] The arrows appearing in the microchannels shown in FIGS. 2A, 2Band 2C indicate the direction of flow of cells, dilution buffer or otheragent, as the case may be, along the flow path within the microchannel.

[0038] It should be clear that under appropriate conditions, cells maybe lysed using only a chemical lysing agent added through channel(s) 85a and/or 85 b, with subsequent chemical separation, e.g. electrophoresisor chromatography, taking place in the cell lysis region, 87.

[0039] In addition to cellular analysis, it would be possible to performother types of analyses using this type of structure. An example wouldbe the analysis of compounds that are produced by solid phase synthesison beads made of materials such as polystyrene. It is possible to designcovalent linkers that hold the compounds on the beads such that theywill be released when exposed to appropriate reagents. In this case sucha chemical additive could be added at the channels 65 (FIG. 2A), 75 a(FIG. 2B) or 85 a, 85 b (FIG. 2C) to release the bead bound o compounds.Subsequent analysis by various techniques including chemicalseparations, flow injection analysis, or mass spectrometry afterelectrospray ionization could then be performed.

[0040]FIGS. 3A and 3B illustrate microchannel structures in which achange in applied electric field strength within a microchannel iseffected by varying a cross-sectional dimension of the microchannel. Theembodiments shown in FIGS. 3A and 3B are similar in that eachmicrochannel structure 110, 120 includes a pair of electrodes 112, 116and 122, 126 positioned at spaced apart sites along microchannels 114,124, each electrode being connected to a source of electric potential119, 129. In this way, a cell lysis region 117, 127 is establishedwithin the electric field applied between the electrodes. In FIG. 3A, aportion of microchannel 114 is expanded relative to the remainder of themicrochannel, whereas in FIG. 3B, a portion of microchannel 124 isconstricted relative to the remainder of the microchannel. By abruptlyincreasing and then decreasing the channel width, for example, orvice-versa, the electric field strength also changes abruptly,generating sufficient force to lyse the cell.

[0041] The arrows shown in microchannels 114, 124 of FIGS. 3A and 3B,respectively, have the same significance as mentioned above, withreference to FIG. 1A.

[0042] When hydraulic pressure is applied to a microchip device, asdescribed herein, using the same reservoir or other site at which anelectrode is placed, the fluid flow can be adversely affected by theevolution of gas at the electrode. This problem can be overcome byrepositioning the electrode as shown in FIG. 4. For example, electrode132 can be placed at or near the end of auxiliary microchannel 133, asshown in FIG. 4A downstream of intersection 135, or at or near the endof microchannel 148 that is connected downstream of intersection 145 toa portion of microchannel 144 via bridging membrane 151, as shown inFIG. 4B. Any gaseous electrolysis products that may be generated at theelectrode can be vented to atmosphere, e.g., through a reservoir (notshown) in fluid communication with auxiliary channel 133 or channel 148,as the case may be, without interfering with the fluid flow.

[0043] Another embodiment of a microfluidic system embodying the presentinvention is shown in FIG. 5. At least one cell 161 is transportedthrough microchannel 164 by the application of hydraulic pressure to thesystem. The pressure can be super-ambient and applied at channel segment164, 165 a and 165 b, or sub-ambient and applied at channel segment 168.In this latter case, microchannels 164, 165 a, 165 b must beproportioned correctly to obtain the desired flow rates in each channel.This will establish the direction of flow as indicated by the arrow inFIG. 5A. The channels can be fabricated so that most of thecell-containing sample passes from channel segment 164 to channelsegment 168. The side channels 165 a and 165 b may be fabricated so thatthe resistance to hydro-dynamic flow (R) is substantially higher thanthat of channel segment 164. Flow resistance (R) can be controlled byadjusting the length of the channel segment and/or its cross-sectionalarea. The flow resistance can be made much larger for the side channelsthan for microchannel segment 164. An electric field is applied betweenchannel segments 165 a and 165 b, as shown in FIG. 5A. This field may beDC, AC, pulsed or a combination thereof. Pressure driven flow can beused to transport the cell to intersection 163, as seen in FIG. 5B.Lysis is induced by the applied electric field and causes some of thecell lysate to form analyte plug 171 which electromigrates into one ofthe side channels, as seen in FIG. 5C. The analyte plug can be made topass through microchannel segments 165 a and/or 165 b, depending uponwhether electrosmotic flow is present and the nature of the electriccharges of the species present in the cell lysate. The analyte plug canbe separated into discrete segments 173 a, 173 b, 173 c based uponchemical differences in the side channel. These analyte segments can bedetected at some point downstream of the intersection.

[0044] The presence of cell 161 in the intersection 163 could bedetected by a change in conductivity and can be used to trigger eventssuch as the application of a cell lysis electric field or otherprocedures, as previously described.

[0045] With the appropriate pressures and voltages applied, the cellulardebris 195, including the cell membrane and/or cell wall (if present),can be transported down channel segment 168 in FIG. 5D after cell lysis.This helps prevent the cellular debris from accumulating in sidechannels 165 a and 165 b and interfering with the transport of analyteplug 171. Channel segment 168 can be expanded in depth and/or width toallow greater area for the accumulation of cellular debris over timefrom multiple cell lysis events. Cellular debris accumulation could alsobe controlled using buffer additives or occasionally flushing channels164 and 168 with a cleaning solution.

[0046] The microfluidic system shown in FIG. 6 combines features of flowcytometry to spatially confine and detect the presence of the cell asdescribed in U.S. Pat. Nos. 5,858,187 and 6,120,666, and the cell lysisfeatures of FIG. 5, described above. The cell-containing fluid sample istransported down microchannel 174 toward channel segment 174 b. Fluidstreams from side channels 175 a and 175 b function to position thecell, preferably in the center of channel segment 174 b. In channelsegment 174 b, the presence of the cell can be detected by scatteredlight, as described above with reference to FIG. 1C. Alternatively, thepresence of a cell entering channel 174b could be detected by monitoringthe conductivity of the solution between channels 175 a and 175 b.Knowledge of a cell transiting channel 174 b toward intersection 176could be used to trigger various events such as previously described,e.g., application of the lysis electric potential, data collection, oradjusting fluid flow in various channels to control cell arrivalfrequency. When the cell arrives in the cell lysis region 176 at theconfluence of channel segment 174 b and side channels 177 a and 177 b,the cell is lysed by applying an electric field in side channels 177 aand 177 b across the cell lysis region 176 similar to the processdescribed above for FIG. 5. A portion of the cell lysate can betransported into side channel 177 a or 177 b depending on the flow rateand electric potential applied. Proper adjustment of the flow velocityin the various channels could allow cellular debris to be transporteddown channel segment 174 c. The cell lysate introduced into channelsegment 177 a or 177 b is then analyzed. If a cell is present in channelsegment 174 b or in the cell lysis region 176 or the intracellularcontents are being analyzed in side channels 177 a or 177 b, thetransport of additional cells into channel segment 174 b can betemporarily stopped by slowing or stopping the flow of cells inmicrochannel 174.

[0047] Similar to discussions above for FIGS. 2A, 2B and 2C, themicrofluidic system described in FIG. 6 could also be used to analyzecompounds bound to solid phase, synthesized beads. A chemical cleavingagent could be added through microchannels 175 a and/or 175 b. When abead traverses from microchannel 174 to 174 b, it is exposed to thechemical cleaving agent thus releasing the bound compounds intomicrochannel 174. A portion of the released compounds can be injectedinto microchannels 177 a and/or 177 b and analyzed by any desiredchemical separation technique and/or flow injection analysis, butpreferably an electromigration technique such as electrophoresis orelectrochromatography.

[0048] In all of the microfluidic systems described herein, the cellsupstream from the cell lysis region are preferably manipulatedhydraulically and are not exposed to electric fields. This is shown inFIG. 1, for example, by the large “X”, which indicates the field freechannel segment through which cells are transported to the cell lysisregion. Hydraulic pressure is the preferred motive force becauseelectric fields tend to disrupt the normal metabolic state of a cell.However, under certain conditions of operation, low electric fieldstrengths may also be used for cell transport and other manipulationwith minimal disruption to normal cell processes. The pressuredifferential employed to create the hydraulic flow, indicated as ΔP inFIGS. 1, 4 and 5 can be generated external to the microfluidic device,using super-ambient or sub-ambient applied pressures, or on the deviceitself, using electrokinetically induced pressures, as described in theaforementioned U.S. Pat. Nos. 6,110,343 or 6,231,737 and U.S. patentapplication Ser. No. 09/244,914.

[0049] Flow cytometry like cellular manipulations of the type used inthis invention are described in greater detail in the aforementionedU.S. Pat. Nos. 5,858,187 and 6,120,666, the entire disclosures of whichare incorporated by reference herein.

[0050] In carrying out the method of the present invention, cells arelysed when they come into contact with the electric field in the celllysis region, due to polarization of the cell membrane which results inrupture of the cell. The electric field can be constant or can vary withtime. If the field changes with time, then the wave form can be appliedcontinuously or pulses can be triggered, for instance by optical means,utilizing light scattering or fluorescence, for example, to detect thecells as they approach or enter the cell lysis region; or detecting achange in conductivity between two electrodes as a cell passes betweenthem. The two electrodes used to detect changes in conductivity may bethe same electrodes with which the electric field is applied to performlysis.

[0051] The frequency, period, duty cycle and wave form type of theelectric field can be varied to optimize the speed and efficiency ofcell lysis. The electric field strength applied for cell lysis willordinarily be in the range of 300-1200 V/cm. The specific electric fieldstrength applied in a particular instance will depend upon the celltype, the presence or absence of a cell wall, the cell orientation inthe electric field, and the cell membrane composition, among otherfactors.

[0052] Downstream of the cell lysis region, the released contents of thecell(s) may be analyzed by, for example, flow injection analysis,electrophoresis, electrochromatography, micellar electrochromatography,chromatography, hydrodynamic chromatography, or molecular sieving.

[0053] When a pulsed or AC field is used, a DC offset can be applied tothe oscillating waveform to maintain an electric field in the channeldownstream of the cell lysis region. This DC offset defines the averageelectric field in the channel segment used for separation and allows theelectrokinetic separations of the cell lysate to continue during theminimum periods of the duty cycle. If the AC frequency of the appliedelectric field is sufficiently high, then the cell lysate will onlyexperience the DC offset of the wave form. Such operating conditionsmight be optimal as the migration times of the analytes, which are usedfor identification, will be more consistent and easier to predict.Another method to generate an effective DC offset is to use a squarewave form centered at zero with a duty cycle≦0.50, where the duty cycleis defined as the time spent at the high potential divided by the totalcycle of time.

[0054] The microfluidic systems and methods of this invention may beused for the manipulation and analysis of a wide range of cells ofpotential scientific interest, including, without limitation, cells,yeasts, bacteria, algae and protists, whether pathogenic or not.Moreover, it may be possible to analyze synthetic particles such asliposomes, vesicles or beads.

[0055] The following examples describe the invention in further detail.These examples are provided for illustrative purposes only, and shouldin no way be construed as limiting the invention.

EXAMPLE I

[0056] A microchip design of the type shown in FIG. 2B has been testedusing the immortal Jurkat T lymphocyte cell line. The cells were labeledwith calcein AM (Catalog #C-1430; Molecular Probes, Eugene, Oreg.) andcontained in PBS. The cross intersection 73 was visualized using a videorate CCD camera (30 frames/sec) and the cells were hydraulicallytransported to the cross intersection by applying sub-ambient pressureto the lower segment of microchannel 74. The cells appear elongated inthe images in FIG. 7 because the fluorescence signal from the calcein inthe cell is integrated over 33 msec for each video frame. The streaklength increases after the cell enters the lower segment of microchannel74 because the flow rate in this channel segment is equal to the sum ofthe flow rates in the upper and side channel segments leading tointersection 73. A pulsed electric field (square wave) was appliedbetween the side channel segments 75 a, 75 b and the lower segment ofmicrochannel 74. At the nadir of the wave 0.35 kV was applied for 0.4second and at the apex 2.6 kV was applied for 0.2 second. Any cell(s)present in intersection 73 or in the segment of microchannel 74 justbelow intersection 73 when the field was applied, were quickly lysed.Lysis was complete within about 66 msec (two video rate frames) leavinga plume of calcein in the channel (FIG. 7A, frames 4 and 5). The axialextent of the analyte plug thus obtained is small compared to thetypical separation column length. Yet it is large enough that theproteolytic enzymes should be sufficiently diluted to minimize theiractivity. Generally, the axial length of the analyte plug will be in therange of 1 to 100 μm, but could be as large as 1 mm.

[0057] A similar experiment with only hydrodynamic flow, i.e., withoutan applied electric potential, is shown in FIG. 7B. The cell was notlysed during the time that it was in the field of view of the videocamera (about 330 msec).

[0058] It was found that adding 25 mM sodium dodecylsulfate to sidechannels 75 a, 75 b helped to prevent the ruptured cell membrane fromadhering to the channel walls downstream from intersection 73.

[0059] For the best separation results it would be advantageous tomaintain a high electric field at all times during operation of themethod. However, the high conductivity of the PBS sample buffer preventsa constant application of a high field because of significant Jouleheating of the sample. Accordingly, a pulsed field (square wave) isrecommended for use with a positive DC offset (about 350 V). Thepositive DC offset allows the electrokinetic separation of the celllysate downstream of intersection 73 to continue during the low portionsof the duty cycle.

EXAMPLE II

[0060]FIG. 8 shows the separation of several fluorescently labeledcomponents released from a single cell utilizing a microchip design ofthe type depicted in FIG. 5, above. These components are Oregon greenand some Oregon green degradation products. To generate this data,living Jurkat cells were loaded with the membrane permeable dye Oregongreen diacetate. The cells 161 metabolized the dye transforming it intoa membrane impermeable form that was trapped in the cell. The cell 161was then transported through microchannel 164 to the cross intersection163, where it was lysed by the electric field applied through channels165 a and 165 b. A portion of the lysed contents in analyte plug 171 wasthen transported by the electric field into side channel 165 b andelectrophoretically separated into multiple analyte segments 173 a, 173b, and 173 c and detected by fluorescence.

[0061] While certain embodiments of the present invention have beendescribed and/or exemplified above, various other embodiments will beapparent to those skilled in the art from the foregoing disclosure. Thepresent invention is, therefore, not limited to the particularembodiments described and/or exemplified, but is capable of considerablevariation and modification without departing from the scope of theappended claims.

What is claimed is:
 1. A method of releasing the intracellular contentsof at least one cell of a cell-containing fluid sample for analysis,said method comprising the steps of: a. providing a substrate having amicrochannel structure which includes at least one microchannel therein;b. generating an electric field from a source of electric potential,said electric field being applied in a spatially defined region of saidat least one microchannel, comprising a cell lysis region, and havingsufficient strength to induce cell lysis; and c. positioning said atleast one cell of said fluid sample in said cell lysis region for a timesufficient to release said intracellular contents of said at least onecell into said fluid sample, thereby providing a volume of analyte insaid at least one microchannel.
 2. The method according to claim 1,wherein said positioning step comprises causing said cell-containingfluid sample to flow into said cell lysis region.
 3. The methodaccording to claim 2, including causing said cell-containing fluidsample to flow into said cell lysis region under the influence ofhydraulic pressure.
 4. The method according to claim 2, includingcausing said cell-containing fluid sample to flow into said cell lysisregion under the influence of electrokinetically-induced pressure. 5.The method according to claim 2, including causing said cell-containingfluid sample to flow into said cell lysis region under the influence ofan electric potential.
 6. The method of claim 1 further includingintroduction of a chemical lysing agent into said cell lysis region. 7.The method of claim 1, wherein the strength of the electric fieldapplied in step b. is substantially constant over time.
 8. The methodaccording to claim 7 further including the steps of detecting a changein conductivity caused by the passage of said at least one cell throughsaid at least one microchannel, and activating said source of electricpotential in response to said detected change in conductivity, therebyto produce a substantially constant electrical field.
 9. The method ofclaim 1, wherein the strength of the electric field applied in step b.varies over time.
 10. The method according to claim 9 further includingthe steps of detecting a change in conductivity caused by the passage ofsaid at least one cell through said at least one microchannel, andactivating said source of electric potential in response to saiddetected change in conductivity, thereby to produce a varying electricalfield.
 11. The method according to claim 9, wherein said electric fieldapplied in step b. is pulsed.
 12. The method according to claim 11further including the steps of detecting a change in conductivity causedby the passage of said at least one cell through said at least onemicrochannel, and activating said source of electric potential inresponse to said detected change in conductivity, thereby to produce apulsed electrical field.
 13. The method according to claim 12,additionally including the step of deactivating said source ofelectrical potential in response to a further change in saidconductivity.
 14. The method according to claim 7, additionallyincluding the steps of directing light at said at least onemicrochannel, detecting scattered light from said at least one cell insaid at least one microchannel, and activating said source of electricalpotential in response to said detected scattered light, thereby toproduce a substantially constant electric field.
 15. The methodaccording to claim 9, additionally including the steps of directinglight at said at least one microchannel, detecting scattered light fromsaid at least one cell in said at least one microchannel, and activatingsaid source of electrical potential in response to said detectedscattered light, thereby to produce a varying electric field.
 16. Themethod according to claim 11, additionally including the steps ofdirecting light at said at least one microchannel, detecting scatteredlight from said at least one cell in said at least one microchannel, andactivating said source of electrical potential in response to saiddetected scattered light, thereby to produce a pulsed electric field.17. The method according to claim 16, additionally including the step ofdeactivating said source of electrical potential in response to anabsence of scattered light.
 18. The method of claim 1, wherein thestrength of the electric field applied in step b. is caused to vary byvarying at least one cross-sectional dimension of the microchannelwithin said cell lysis region.
 19. The method according to claim 2,additionally including the step of causing said fluid sample to flowthrough and beyond said cell lysis region and analyzing said volume ofanalyte in said microchannel structure beyond said cell lysis region.20. The method according to claim 19, wherein said volume of analyte isanalyzed by a technique selected from the group consisting of flowinjection analysis, electrophoresis, chromatography,electrochromatography, micellar electrochromatography, hydrodynamicchromatography, molecular sieving, or a combination thereof, causingseparation of said volume of analyte into discrete segments.
 21. Themethod according to claim 20 further comprising subjecting at least onediscrete segment to further analysis.
 22. The method of claim 21,including the further step of electrospraying said at least one discretesegment for analysis by mass spectroscopy.
 23. The method of claim 2,wherein said electric field is oriented axially with the direction offlow of said cell-containing fluid sample.
 24. The method of claim 2,wherein said electric field is oriented perpendicularly to the directionof flow of said cell-containing fluid sample.
 25. A microfluidic systemfor transport and lysis of at least one cell of a cell-containing fluidsample, said system comprising a source of electric potential and asolid substrate having at least one microchannel with a longitudinalaxis, said microchannel having a first wall portion on one side of saidaxis, a second wall portion on another side of said axis and a celllysis region between said first and second wall portions, a first and asecond electrical contact positioned adjacent said first and second wallportions of said at least one microchannel, said first and secondelectrical contacts being spatially separated by said cell lysis regionand being electrically isolated from one another, said first and secondelectrical contacts being connected to said source of electricalpotential which is operative to apply an electric field to said celllysis region within the microchannel space between said first and secondelectrical contacts, and means for transporting said cell-containingfluid sample along said at least one microchannel.
 26. The microfluidicsystem according to claim 25, wherein said first and second wallportions are on opposite sides of said longitudinal axis.
 27. Themicrofluidic system according to claim 25, wherein said transportingmeans comprises means to apply a superambient hydraulic force throughsaid at least one microchannel upstream of said cell lysis region. 28.The microfluidic system according to claim 25, wherein said transportingmeans comprises means to apply a subambient hydraulic force through saidat least one microchannel downstream of said cell lysis region.
 29. Amicrofluidic system according to claim 25, wherein said first and secondelectrical contacts comprise areas extending longitudinally of said atleast one microchannel, said areas being substantially coextensive inlength with each other and with said cell lysis region.
 30. Amicrofluidic system for transport and lysis of at least one cell of acell-containing fluid sample and separation of the intracellular contentof said at least one cell, said system comprising a solid substratehaving at least one microchannel disposed therein, said microchannelhaving a cell transport segment and a separation segment having firstand second end portions, first and second electrical contacts adjacentsaid microchannel, intermediate said transport segment and saidseparation segment, and spatially separated from one another, themicrochannel space between said electrical contacts defining a celllysis region, said electrical contacts being connected to a source ofelectric potential to apply an electric field to said cell lysis region;means for flowing said cell-containing fluid sample through said atleast one microchannel; and means between said first and secondseparation segment end portions for effecting separation of saidintracellular contents of said at least one cell.
 31. The microfluidicsystem according to claim 30, wherein the cross-sectional area of atleast a portion of said at least one microchannel within said cell lysisregion is different from the cross-sectional area of at least a portionof the remainder of said at least one microchannel.
 32. The microfluidicsystem according to claim 31, wherein the cross-sectional area of atleast a portion of said at least one microchannel within said cell lysisregion is constricted relative to said cross-sectional area of at leasta portion of the remainder of said at least one microchannel.
 33. Themicrofluidic system according to claim 31, wherein the cross-sectionalarea of at least a portion of said at least one microchannel within saidcell lysis region is expanded relative to said cross-sectional area ofat least a portion of the remainder of said at least one microchannel.34. The microfluidic system according to claim 25 further comprising:means for sensing the presence of cell in said microchannel; and meansresponsive to said sensing means for triggering an operational event.35. The microfluidic system according to claim 34, wherein theoperational event is selected from the group consisting of: applicationof a cell lysis electric field in said cell lysis region, recordation ofa time mark to indicate the start of a separation process, adjustment offluid flow to cell arrival frequency, or a combination thereof.
 36. Themicrofluidic system according to claim 34, wherein said sensing meanscomprises third and fourth electrical contacts positioned adjacent tosaid first and second wall portions upstream of said cell lysis region.37. The microfluidic system according to claim 34, wherein said sensingmeans comprises an optical probe.
 38. The microfluidic system accordingto claim 37 wherein said optical probe comprises: a light source adaptedfor directing light into said microchannel; and a light detector adaptedfor detecting light scattered by a cell in said microchannel.
 39. Amethod of releasing and analyzing compounds from at least one particleof a particle-containing fluid sample, comprising the steps of: a.providing a substrate having a microchannel structure which includes afirst microchannel intersecting with a second microchannel at anintersection; b. positioning a particle of a particle-containing fluidsample in said intersection, said particle containing at least onecompound; c. introducing a chemical lysing solution into saidintersection through said second channel, whereby said at least onecompound is released into the fluid sample, thereby providing a volumeof analyte in said intersection; d. causing said fluid sample containingsaid analyte to flow through and beyond said intersection; and then e.analyzing said volume of analyte in said first microchannel beyond saidintersection.
 40. The method according to claim 40, wherein the particleis cell.
 41. The method according to claim 40, wherein the particle is asynthetic particle selected from the group consisting of liposomes,vesicles, beads, or a combination thereof.
 42. The method according toclaim 41, wherein the particle is a solid that has the compoundchemically bonded to the surface thereof.